Urinary tract infection (UTI) is the second most common infection in humans after those involving the respiratory tract. This results not only in huge annual economic costs, but in decreased workforce productivity, and high patient morbidity. At least 80% of these infections are caused by uropathogenic Escherichia coli (UPEC). Antibiotic treatment is generally effective for eradication of the infecting strain, however, documentation of increasing antibiotic resistance, allergic reaction to certain pharmaceuticals, alteration of normal gut flora, and failure to prevent recurrent infections, represent significant barriers to treatment. As a result, approaches to prevent UTI such as vaccination represent a gap that must be addressed. Excellent progress (36 publications) has been made in the previous funding period developing a preventive vaccine against UPEC. The long-term research goal is to prevent UTIs in women with recurrent UTI. The objective during this funding period is to identify the optimal combination of protective antigens for inclusion in n effective UTI vaccine, optimal adjuvant, optimal dose, and optimal route of delivery. The central hypothesis states that a multi-subunit vaccine elicits antibody that protects against experimental challenge with UPEC strains. The rationale for the proposed work is to protect women from the development of UTI by administration of a preventive vaccine. The central hypothesis will be tested and objectives will be completed by carrying out two specific aims: 1) Combine protective antigens to establish an effective multi-subunit vaccine to prevent urinary tract infection. 2) Identify the mechanism of protection for the optimized vaccine against uropathogenic E. coli in urinary tract infection. In the last funding period, we systematically identified four antigens tha can individually protect experimentally infected mice from colonization of the bladder and/or kidneys by UPEC when administered intranasally with cholera toxin (CT) as adjuvant. To advance the vaccine for utility in humans, we will group the individual antigens (IreA, Hma, IutA, FyuA) into an effective combination to establish a multi-subunit vaccine, and optimize the adjuvants, doses, and routes of inoculation currently used for immunization of humans. We demonstrated for all four vaccine antigens that antigen-specific serum IgG represents a strong correlate of protection in vaccinated mice. High antibody titers correlate with low CFUs of UPEC following transurethral challenge of vaccinated mice. Fundamental unanswered questions regarding the role of antigen-specific antibodies in protection afforded by the vaccine will be pursued including: assessment of long-term protection, passive immunization, the involvement of B cells in protection, and whether antibodies bind to the surface of UPEC and inhibit iron acquisition and uptake. Sera from women with and without histories of UTI will be tested for antibody levels to vaccine antigens, epitope recognition, and the potential for eliciting opsonophagocytosis of UPEC. At the conclusion of the funding period, our expected result is an optimized vaccine ready for the next phase of development to prevent UTI in women with recurrent UTI.